Inaccuracies in biochemically characterizing the amount and CO-fixing properties of the photosynthetic enzyme Ribulose-1,5-bisphosphate (RuBP) carboxylase/oxygenase continue to hamper an accurate evaluation of Rubisco mutants selected by directed evolution. Here, we outline an analytical pipeline for accurately quantifying Rubisco content and kinetics that averts the misinterpretation of directed evolution outcomes. Our study utilizes a new T7-promoter regulated Rubisco Dependent (RDE3) screen to successfully select for the first Rubisco (Rubisco) mutant with improved CO-fixing properties. The Rubisco contains four amino acid substitutions in the large subunit (RbcL) and an improved carboxylation rate (, up 27%), carboxylation efficiency (/ for CO, increased 17%), unchanged CO/O specificity and a 40% lower holoenzyme biogenesis capacity. Biochemical analysis of Rubisco chimers coding one to three of the altered amino acids showed Lys-83-Gln and Arg-252-Leu substitutions (plant RbcL numbering) together, but not independently, impaired holoenzyme (LS) assembly. An N-terminal Val-11-Ile substitution did not affect Rubisco catalysis or assembly, while a Tyr-345-Phe mutation alone conferred the improved kinetics without an effect on Rubisco production. This study confirms the feasibility of improving Rubisco by directed evolution using an analytical pipeline that can identify false positives and reliably discriminate carboxylation enhancing amino acids changes from those influencing Rubisco biogenesis (solubility).